All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Inflammation is the body's response to injury, infection or to molecules perceived by the immune system as foreign. Absent, excessive or uncontrolled inflammation results in a vast array of diseases such as asthma, arthritis and autoimmune diseases, adult respiratory distress syndrome (ARDS), cardiovascular inflammation and gastrointestinal inflammation. Numerous studies have demonstrated the participation of primed neutrophils, monocytes and macrophages in such inflammatory diseases. More recently, the role of superoxides release by microglia cells in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS) as well as brain ischemic and traumatic injury has also been documented.
The production of superoxides by the phagocyte NADPH oxidase and pro inflammatory lipid mediators by phospholipase A2 are among the most important functions for host defense. However, during altered physiological states, superoxides and lipid mediators promote inflammatory reactions and participate in processes that lead to tissue injury and the pathophysiology of various inflammatory diseases. Nowadays non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most widely prescribed drugs for the treatment of inflammatory conditions. However, they present unwanted side effects, the most common being ulceration and bleeding in the gastrointestinal tract. Moreover, these drugs reduce only the production of prostaglandins and do not affect the production of leukotrienes which have a pivotal role in the recruitment of neutrophils to the site of inflammation. Thus, the search for new anti-inflammatory drugs with fewer side effects continues. Numerous trials have been conducted with agents that block the inflammatory cascade, like corticosteroids, antiendotoxin antibodies, TNF antagonists, IL-1 receptor antagonists and other agents, without significant success.
The present inventor has developed a cell line, stable clones of PLB-985 cells lacking the expression of cytosolic phospholipase A2 (cPLA2), and demonstrated that cPLA2, in addition to its known role in the production of pro-inflammatory lipid mediators, is essential for activation of the phagocyte NADPH oxidase complex after its assembly. The association between these two enzymes provides the molecular basis for activation of the assembled NADPH oxidase by arachidonic acid (AA) released by cPLA2. [Dana, R. et al. (1998) J. Biol. Chem. 273:441-5; Lowenthal, A. and Levy, R. (1999) J. Biol. Chem. 274: 21603-10; Levy, R. et al. (2000) Blood. 95:660-5; Pessach, I. et al. (2001) J. Biol. Chem. 276:33495-503; Shmelzer, Z. et al. (2003) J. Cell Biol. 162:683-692; Tarsi-Tsuk, D. and Levy, R. (1990) J. Immunol.; 144:2665-2670; Dana, R. et al. (1994) Biochem J. 297:21′7-223; Hazan-Halevy, I. et al. (2000) J. Biol. Chem. 275:12416-12423]. Since cPLA2 is required for oxidase activation, its inhibition should not only diminish the formation of inflammatory mediators, but should also regulate the uncontrolled accelerated release of oxygen radicals that participate in the pathogenesis of inflammatory diseases. Moreover, the inventor's studies have shown that during inflammation in vivo or inflammatory conditions in vitro, the level and activity of both cPLA2 and NADPH oxidase enzymes are elevated, in neutrophils and monocytes [Levy, R. et al. (1994) Biochim. Biophys. Acta 1220:261-265; Shaked, G. et al. (1994) J. Trauma 37:22-29; Levy, R. et al. (2000) Blood 95:660-665; Levy, R. and Malech, H. (1991) J. Immunol. 147:3066-3071; Levy, R. et al. (1994) Biochim. Biophys. Acta 1220:253-260; Reizenberg, K. et al. (1997) Eur. J. Clin. Invest. 27:398-404]. Surprisingly, a recent report described that addition of cPLA2 inhibitor pyrrolidine to neutrophils did not inhibit NADPH oxidase activity [Rubin, B. B. et al. (2005) J. Biol. Chem. 280:7519-29], however this effect might have been due to the methodology applied, which did not allow sufficient accumulation of the drug in the neutrophils (data not shown). Although methods of treating inflammatory conditions by inhibiting cPLA2 have been described, they involved the use of substances like trifuoromethylketone (TFMK), causing dose-dependent attenuation of airway inflammation [US Patent Application No. 20020165119, USSN 062730], or indole compounds, which inhibited various forms of PLA2 [U.S. Pat. No. 6,797,708], but no inhibitor unique to cPLA2 has been described for treatment of inflammation to date. Currently, potent cytosolic PLA2 inhibitors are not available for clinical use in human or animals. All inhibitors against cPLA2 so far were engineered to compete with the substrate. Since all types of PLA2 cleave the fatty acid from the sn-2 position of phospholipids, they are also inhibited by the same inhibitors (although some times with lower efficiency). Although several compounds were described as specific inhibitors of cPLA2, they were found to also inhibit other PLA2 enzymes and vice versa. Because of the lack of specific inhibitor for each PLA2 subtype, the antisense technology provides an effective approach to inhibit, a specific type of PLA2. Indeed, the results presented herein suggest that a drug targeted directly to cPLA2 will specifically inhibit cPLA2 activity. Moreover it also results in the regulation of both cPLA2 and NADPH oxidase to produce pro-inflammatory mediators and superoxides.
Antisense oligonucleotides targeted against the cPLA2 mRNA sequence have been reported in the past as capable of inhibiting cPLA2 transcript expression [U.S. Pat. No. 6,008,344]. However, these oligonucleotides did not demonstrate inhibition of cPLA2 protein expression, and were introduced into cells in the presence of lipofectin.
In addition, three other antisense oligonucleotides targeted to cPLA2 have been described; P1 (Table 1, SEQ. ID. No. 8) [Roshak, A. (1994) J. Biol. Chem. 269(42): 25999-26005; Muthalif, M. M. et al. (1996) J. Biol. Chem. 271(47): 30149-30157; Marshall, L. (1997) J. Biol. Chem. 272(2): 759-765; Anderson, K. M. et al. (1997) J. Biol. Chem. 272(48): 30504-30511]; P2 (Table 1, SEQ. ID. No. 9) [Li, Q. and Cathcart, M. K. (1997) J. Biol. Chem. 272(4): 2404-2411; Zhao, X. et al. (2002) J. Biol. Chem. 277(28): 25385-25392]; and P3 (5′-GTGCTGGTAAGGATCTAT-3′; SEQ. ID. No. 12) [Locati, M. (1996) J. Biol. Chem. 271(11): 6010-6016], mainly evaluating the effect of inhibiting cPLA2 in smooth muscle cells and human monocytes function. P1 was used together with lipofectin. P1 and P2, with phosphorothioate modifications in all bases, had a significant effect only when used at 5 μM, which the present inventor found to be toxic to the cells. P3 was used at 10 μM (or even higher concentration, 10 times higher than what was used by the present inventor).
Thus, it is an object of the present invention to provide novel antisense oligonucleotides against the cPLA2 mRNA, and their use in the inhibition of cPLA2 expression and superoxide production, in order to inhibit pro-inflammatory processes. Consequently, the antisense oligonucleotides claimed in the present invention are also sought as anti-inflammatory agents.
Other uses and objects of the invention will become clear as the description proceeds.